RNA interference (RNAi) is an evolutionarily conserved, sequence specific mechanism triggered by double stranded RNA (dsRNA) that induces degradation of complementary target single stranded mRNA and “silencing” of the corresponding translated sequences (McManus and Sharp, Nature Rev. Genet. 3:737 (2002)). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active “short-interfering RNA” (siRNA) sequences of about 21-23 nucleotides in length (Elbashir, et al., Genes Dev. 15:188 (2001)).
siRNA can be used downregulate or silence the transcription and translation of a gene product of interest. For example, it is desirable to downregulate genes associated with liver diseases and disorders such as hepatits. In particular, it is desirable to down-regulate genes associated with hepatitis viral infection and survival.
An effective and safe nucleic acid delivery system is required for interference RNA to be therapeutically useful. Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of limitations, such as the potential for reversion to the wild type as well as immune response concerns. As a result, nonviral gene delivery systems are receiving increasing attention (Worgall, et al., Human Gene Therapy 8:37 (1997); Peeters, et al., Human Gene Therapy 7:1693 (1996); Yei, et al., Gene Therapy 1: 192 (1994); Hope, et al., Molecular Membrane Biology 15:1 (1998)). Furthermore, viral systems are rapidly cleared from the circulation, limiting transfection to “first-pass” organs such as the lungs, liver, and spleen. In addition, these systems induce immune responses that compromise delivery with subsequent injections.
Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Felgner, Scientific American 276:102 (1997); Chonn, et al., Current Opinion in Biotechnology 6:698 (1995)). For instance, cationic liposome complexes made of an amphipathic compound, a neutral lipid, and a detergent for transfecting insect cells are disclosed in U.S. Pat. No. 6,458,382. Cationic liposome complexes are also disclosed in U.S. Patent Publication No. 2003/0073640.
Cationic liposome complexes are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques 19:816 (1995); Li, et al., The Gene 4:891 (1997); Tam, et al, Gene Ther. 7:1867 (2000)). As large, positively charged aggregates, lipoplexes are rapidly cleared when administered in vivo, with highest expression levels observed in first-pass organs, particularly the lungs (Huang, et al., Nature Biotechnology 15:620 (1997); Templeton, et al., Nature Biotechnology 15:647 (1997); Hofland, et al., Pharmaceutical Research 14:742 (1997)).
Other liposomal delivery systems include, for example, the use of reverse micelles, anionic and polymer liposomes. Reverse micelles are disclosed in U.S. Pat. No. 6,429,200. Anionic liposomes are disclosed in U.S. Patent Application No. 2003/0026831. Polymer liposomes, that incorporate dextrin or glycerol-phosphocholine polymers, are disclosed in U.S. Patent Application Nos. 2002/0081736 and 2003/0082103, respectively.
A gene delivery system containing an encapsulated nucleic acid for systemic delivery should be small (i.e., less than about 100 nm diameter) and should remain intact in the circulation for an extended period of time in order to achieve delivery to affected tissues. This requires a highly stable, serum-resistant nucleic acid-containing particle that does not interact with cells and other components of the vascular compartment. The particle should also readily interact with target cells at a disease site in order to facilitate intracellular delivery of a desired nucleic acid.
Recent work has shown that nucleic acids can be encapsulated in small (about 70 nm diameter) “stabilized nucleic acid-lipid particles” (SNALP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271 (1999)). These SNALPs typically contain the “fusogenic” lipid dioleoylphosphatidylethanolamine (DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence of a poly(ethylene glycol) (PEG) coating. SNALP have systemic application as they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially at distal tumor sites due to the enhanced vascular permeability in such regions, and can mediate transgene expression at these tumor sites. The levels of transgene expression observed at the tumor site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing plasmid DNA-cationic liposome complexes (lipoplexes) or naked DNA.
Thus, there remains a strong need in the art for novel and more efficient methods and compositions for introducing nucleic acids, such as interfering RNA, into cells. In addition, there is a need in the art for methods of treating or preventing disorders such as hepatitis by downregulating genes associated with viral infection and survival. The present invention addresses this and other needs.